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Production of Fumonisin B and C Analogues by Several Fusarium Species VIKASH SEWRAM,*,† NDUMISO MSHICILELI,†,‡ GORDON S. SHEPHARD,† HESTER F. VISMER,† JOHN P. RHEEDER,† YIN-WON LEE,§ JOHN F. LESLIE,| WALTER F. O. MARASAS†
AND
PROMEC Unit, Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa; School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, Korea; and Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502
Six strains of Fusarium verticillioides, two of F. oxysporum, one strain of F. proliferatum, and a strain of an unidentified species were cultured on maize patties and rice and evaluated for their ability to simultaneously produce fumonisin B (FB) and C (FC) series analogues. Fumonisins were quantified by LC-MS-MS using positive ion electrospray ionization. FC1 provided characteristic fragment ions at m/z 690, 672, 654, 532, 514, and 338 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone, while FC3 and FC4 provided equivalent product ions 16 and 32 amu lower than the corresponding FC1 fragments, respectively. All isolates cultured on maize produced FC4. All isolates except for that of F. proliferatum also produced FC1, and three of the six strains of F. verticillioides produced FC3. All isolates except those of F. oxysporum produced detectable amounts of FB1, FB2, and FB3. Isolates that produced fumonisin B analogues produced at least 10 fold more of the B series analogues than they did of the C series analogues. The results confirm that at least some strains of F. oxysporum produce FC, but not FB, fumonisin analogues and also suggest that the genetics and physiological regulation of fumonisin production may be more complicated than previously envisaged since some strains of F. verticillioides and F. proliferatum as well as the strain of the unidentified species can simultaneously produce both FB and FC analogues. KEYWORDS: Fusarium; fumonisins; tandem mass spectrometry; DNA; amplified fragment length polymorphisms
INTRODUCTION
Fusarium species are important fungal contaminants in agricultural crops that produce a variety of secondary metabolites, including the fumonisins, that are potential risks to human and animal health. Fumonisins (1) are a group of structurally related mycotoxins produced by Fusarium Verticillioides (Sacc.) Nirenberg, formerly known as F. moniliforme Sheldon (2), and related species. Twenty-eight structural fumonisin analogues are known (3), but most research has focused on the most widespread natural forms, which are members of the B-series (FB), for example, fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3). The fumonisins have been shown to affect several target tissues in animals resulting in disease syndromes such as equine leukoencephalomalacia and porcine pulmonary edema as well as causing nephrotoxicity, hepato* Corresponding author. Tel: +27-21-938 0272; fax: +27-21-938 0260; e-mail:
[email protected]. †PROMEC Unit. ‡ Present address: Woolworths Food Chemistry Laboratory, 93 Longmarket Street, Cape Town, 8001, South Africa. § Seoul National University. | Kansas State University.
toxicity, and hepatocellular carcinoma in rats (4). The consumption of fumonisin-contaminated corn has been statistically associated with the high incidence of esophageal cancer in rural areas of South Africa (5) and China (6). In addition to the B-series fumonisins (FB), related compounds belonging to the A-, P-, and C-series also have been described. The A-series fumonisins (FA) are N-acetyl analogues of the B-series and have little or no biological activity (7). The P- and C-series of fumonisins differ from the B-series in that the fumonisin P analogues have a 3-hydroxypyridinium functional group in place of the C-2 amine group (8) and the fumonisin C analogues lack a terminal methyl group at C-1 (Figure 1). Fumonisin C1 (FC1) was the first fumonisin C analogue to be characterized (9) and was isolated from a culture of “F. moniliforme” (FRC M-2326) that originated on maize from Maryland (Jean Juba, Fusarium Research Center, Pennsylvania State University, personal communication, October 8, 2003). Fumonisins C3 (FC3) and C4 (FC4) subsequently were detected from strains (KSU A-00816, KSU A-00817, and KSU A-00819) first identified as F. moniliforme from maize in South Carolina (10, 11) and F. oxysporum Schlecht. emend Snyd. &
10.1021/jf050307n CCC: $30.25 © 2005 American Chemical Society Published on Web 05/10/2005
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Figure 1. Chemical structures of the fumonisin B and C analogues investigated in this study.
Hans (12, 13) isolated from carnation and asparagus in Korea. N-acetyl derivatives of fumonisin C analogues also have been reported from wheat cultures of one of the Korean strains of F. oxysporum (13). The natural occurrence of the fumonisin C analogues has not been widely tested, but Seo and Lee (14) reported both their natural occurrence and co-occurrence with fumonisin B analogues in samples of moldy maize from Korea. The FC and FP fumonisins are phytotoxic to duckweed (Lemna pausicotata L.) and cytotoxic to mammalian cell lines H4TG and MDCK (15). FC1, FC2, and hydroxy-FC1 had phytotoxic effects similar to those caused by FB1 or AAL-toxin, indicating that the C-1 terminal methyl group is not required for this biological activity. FC3 and FC4 were less phytotoxic than FC1 and FC2, and FP1 was even less phytotoxic than FC3 or FC4. The cytotoxicity of these analogues to cell lines paralleled their phytotoxicity toward duckweed (15). Our objectives in this study were to confirm earlier reports of fumonisin C analogue production by F. oxysporum (12, 13), to describe additional strains or species producing fumonisin C analogues, and to confirm whether the FB and FC fumonisins are synthesized simultaneously by the same strain of Fusarium Verticillioides, an observation also reported previously by Branham and Plattner (9). MATERIALS AND METHODS Reagents and Standards. Acetonitrile, HPLC grade (>99.9% purity), was purchased from Romil (Cambridge, U.K.). Formic acid (analytical grade) was obtained from Merck (Darmstadt, Germany). Water was purified in a Milli-Q system (Millipore, Bedford, MA). Fumonisin B standards were isolated in the PROMEC Unit by the method of Cawood et al. (16), and fumonisin C standards were prepared by Prof. Y.-W. Lee as described by Seo et al. (13). Fusarium Strains. Two Korean strains of Fusarium oxysporum, CAR (MRC 7547 ) KSU X-12910) and KCTC 16654 (MRC 8184 ) KSU X-12915), have been reported to produce fumonisin C analogues (13, 14). MRC 7547 was isolated from carnation wilt and produces FC1, FC3, FC4, and hydroxy-FC1 in cultures grown on wheat (12). MRC 8184 was isolated from asparagus wilt and produces an isomer of FC1 (iso-FC1) and three N-acetyl derivatives of hydroxy-FC1 (13). Two other putative F. oxysporum strains from Korea, F. oxysporum No. 4 (MRC 8171 ) KSU D-12913) from sesame seeds and F. oxysporum No. 53 (MRC 8172 ) KSU X-12914) from red beans, have not been previously described but can produce moniliformin (Y.-W. Lee, School of Agricultural Biotechnology and Center for Agricultural Biomaterials,
Sewram et al. Seoul National University, October 11, 2003, unpublished). F. moniliforme 94-cob-01 was isolated from corn in Korea that contained more FC1 than FB1. Two single-conidial isolates, MRC 8169 ()KSU A-12911) and MRC 8170 ()KSU A-12912), were obtained from different sectors of the original culture. The South African F. Verticillioides strains, MRC 826, 4315, 4319, and 4321, were isolated from home-grown corn in Centane, Eastern Cape, South Africa (7). These isolates can produce FB2, FB3, and high levels of FB1 (17). These strains are all sexually cross-fertile with the mating type testers for Gibberella moniliformis (Gibberella fujikuroi mating population A) (11). DNA Analyses and Comparisons. Strains for DNA extraction were cultured by inoculating approximately 1 mL of a spore suspension (typically 106-107 conidia) into 40 mL of liquid complete media (18). Isolates were grown on a rotary shaker (150 rpm) for 2 days at room temperature (23-26 °C) and were harvested by filtration through milk filters (KenAG, Ashland, OH). Mycelia were blotted dry between paper towels and the dried mycelia were stored at -20 °C until DNA extraction. DNA was extracted with a cetyltrimethylammonium bromide (CTAB) protocol as described in Kere´nyi et al. (19). All buffers and DNA modifying enzymes were used following either the manufacturer’s instructions or standard protocols (20). Amplified fragment length polymorphisms (AFLPs) were generated with primer pairs EcoRI+TT and MseI+AC used in the final amplifications as described by Zeller et al. (21). EcoRI primers used in the final specific PCR amplifications were labeled with 33γP-ATP. The PCR machine used was a PTC-2000 Thermal Cycler (MJ Research, Inc., Watertown, Massachusetts). The presence or absence of polymorphic AFLP bands ranging in size from 100 to 800 bp in each gel was scored manually and the data were recorded in a binary format. All polymorphic markers in this size range were scored, even those that were unique to a single individual. Bands appearing at the same mobility in different individuals were assumed to represent the same allele. Each band of differing mobility was treated as a single independent locus with two alleles (present or absent), and unresolved bands or missing data were scored as ambiguous. We amplified and sequenced portions of the DNA sequences for β-tubulin, translation elongation factor 1R, and the ribosomal internally transcribed spacer (ITS) region. For the β-tubulin gene, we used PCR primers T1 and T2 and the PCR reaction conditions described by O’Donnell and Cigelnik (22), except that we increased the annealing temperature to 58 °C, to amplify a homologous region of the β-tubulin gene. For translation elongation factor 1R gene, we used primers EF1 and EF2 (23) and an amplification program of 60 s at 94 °C followed by 34 cycles of 58 °C for 60 s, 72 °C for 60 s, and 94 °C for 30 s and then held at 4 °C. For the ITS region, we used primers ITS4 and ITS5 (24) with the same amplification program as for the transcription elongation factor 1R gene except that the annealing temperature was decreased to 52 °C. PCR products were purified and desalted with the Wizard PCR Preps DNA Purification System (Promega, Madison, WI). DNA sequences were obtained by direct sequencing of the PCR products by using the same PCR primers as used for the amplifications from the genomic DNA with an ABI 3700 automated sequencer at the Kansas State University sequencing facility. Sequences from both strands were aligned to minimize ambiguous nucleotide positions and each sequence was then aligned with ClustalW (25), as implemented in the program BioEdit v4.7.8 (26). Preliminarily aligned sequences were further aligned and edited manually with BioEdit. Culture Conditions. Six F. Verticillioides isolates, four from South Africa (MRC 826, 4315, 4319, and 4321) and two from Korea (MRC 8169 and MRC 8170), two confirmed Korean F. oxysporum strains (strain CAR ) MRC 7547 and KCTC 16654 ) MRC 8184), and the two putative Korean F. oxysporum strains (MRC 8171 and MRC 8172) were evaluated. All isolates were cultured on maize patties (300 per strain) consisting of ground whole yellow maize (30 g) mixed with 30 mL distilled water placed in 90-mm Petri dishes and autoclaved at 121 °C for 1 h on two consecutive days. Patties were inoculated with 1 mL of a spore suspension made from lyophilized cultures (1:100 distilled water dilution per 2 mL lyophilized culture vial) and were incubated in the dark at 25 °C for 21 days. The patty cultures were dried at 50
Production of Fumonisin Analogues °C for 24 h, ground to a meal, and then stored at 4 °C until analyzed for fumonisins in triplicate. The four South African F. Verticillioides strains also were grown on rice. Rice cultures consisted of polished rice (1 kg) soaked overnight in distilled water (245 mL), with occasional shaking. Portions of 4050 g were placed in 250-mL Erlenmeyer flasks and were autoclaved for 20 min at 121 °C. The rice cultures were inoculated, incubated, and then processed in the same manner as were the cultures grown on maize patties. Sexual crosses were made with standard mating type tester strains of Gibberella moniliformis, FGSC 7600 (MATA-1) and FGSC 7603 (MATA-2); Gibberella fujikuroi, FGSC 8931 (MATC-1) and FGSC 8932 (MATC-2); and Gibberella intermedia, FGSC 7615 (MATD-1) and FGSC 7614 (MATD-2) available from the Fungal Genetics Stock Center (University of Missouri, Kansas City). Crosses were made on carrot agar by using standard crossing procedures as described by Klittich and Leslie (27). Determination of Fumonisins. Sample extracts from the cultures were prepared by the method of Sydenham et al. (28). In brief, culture material (5 g) from each strain in 100 mL methanol/water (3:1, v/v) was homogenized for 3 min using a Polytron homogenizer (Kinematica, Luzern, Switzerland). Extracts were centrifuged at 4 °C and 4000g for 10 min, filtered (Whatman No. 4), and the filtrate adjusted to pH 6.0 with 1 M NaOH. The solution (10 mL) cleanup was performed on a Bond Elut SAX solid-phase extraction cartridge (500 mg sorbent) (Varian, Harbor City, CA). The eluate from the SAX cartridge was evaporated to dryness at 60 °C under nitrogen, and the residue was redissolved in the mobile phase prior to injection. HPLC-MS Conditions. The HPLC system consisted of a SpectraSERIES P2000 pump and an AS 1000 autosampler (Thermo Separation Products Inc., Riviera Beach, FL) with a 20-µL injection loop. Reversed-phase HPLC was performed using a binary gradient at a flow rate of 0.7 mL/min on a Luna C18 column (4.6 × 150 mm, 5 µm ODS2) (Phenomenex, Torrance, CA). The mobile phase consisted of mixtures of water/acetonitrile/formic acid in the ratio 90:10:0.1 v/v (solvent A) and 10:90:0.1 v/v (solvent B). The initial composition of 80:20 A:B was adjusted linearly over a 35-min period to 65:35 A:B and held for 1 min. The composition was adjusted back to 80:20 A:B in 2 min and retained for the last 2 min. Fumonisin analogues were detected on the basis of their retention times. Positive ion electrospray ionization (ESI) mass spectrometry was performed with a Finnigan MAT (San Jose, CA) LCQ ion-trap mass spectrometer. The MS parameters were optimized separately by direct infusion of each fumonisin B and C standard (1 mg/mL) into the source at a flow rate of 5 µL/min. The use of appropriate segments during the chromatographic run made scanning at optimum conditions possible for each of the toxins. Capillary and source voltages were 3 V and 4.5 kV, respectively, with the heated capillary temperature maintained at 220 °C. Nitrogen was used as both sheath and auxiliary gas (80 and 20 arbitrary units, respectively). Mass spectrometry experiments were initially carried out by scanning from m/z 190 to 730 to detect the protonated molecular ion signals for FB1, FB2, FB3, FC1, FC3, and FC4 at m/z 722, 706, 706, 708, 692, and 676, respectively. MS-MS experiments were subsequently undertaken through collision-induced dissociation (34% collision energy) of the protonated molecular ions to yield fingerprint profiles for each of the toxins. Toxin levels were quantified (limit of detection ) 2 mg/kg) by comparing the resultant peak areas from the extracts with a calibration plot of the standards. RESULTS
Identification of Korean Fusarium Strains. MRC 7547 and MRC 8184 are F. oxysporum on the basis of morphological characters. On the basis of AFLP fingerprint patterns, the two strains are clones (identity at 72/72 AFLP bands), even though they originated from different hosts. The AFLP fingerprints are not particularly similar to those of other known strains of F. oxysporum, for example, KSU nos. X-11391 (40% identity, 39/ 97 matches) and X-11394 (44% identity, 39/89 matches). On the basis of DNA sequences of portions of the genes encoding
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β-tubulin (GenBank no. AY627278), translation elongation factor 1R (GenBank no. AY627279), and the ITS region of the ribosomal DNA repeat unit data (GenBank no. AY627280), these strains also belong to F. oxysporum. F. oxysporum No. 4 (MRC 8171) is not F. oxysporum on morphological criteria but is F. proliferatum (Matsushima) Nirenberg and is cross-fertile (MAT-1) with standard mating type testers of G. intermedia. The AFLP fingerprint is somewhat similar to that of one of the standard mating type strains of F. proliferatum, FGSC (KSU4853) (44% identity). On the basis of DNA sequences of portions of the genes encoding β-tubulin (GenBank no. AY660013), translation elongation factor 1R (GenBank no. AY660014), and the ITS region of the ribosomal DNA repeat unit data (GenBank no. AY660015), this strain also is F. proliferatum. F. oxysporum No. 53 (MRC 8172) is not F. oxysporum on morphological criteria but is Fusarium nygamai. The standard mating type testers of G. nygamai were not cross-fertile with one another, so this strain was not tested with them for crossfertility. MRC 8172 was not cross-fertile with the standard testers of either G. fujikuroi or G. intermedia. The AFLP fingerprints differ significantly from those of mating type standards of F. nygamai (G5111, 19% similarity) and F. proliferatum (D4853, 31% similarity) and most closely resemble those of F. fujikuroi (C1993, 55% similarity). The identity of MRC 8172 as F. fujikuroi is supported by its ITS (Genbank no. AY662329) and β-tubulin (Genbank no. AY662327) sequences. The translocation elongation factor 1R (Genbank no. AY662328) sequence is considerably more distant from other available sequences, for example, Genbank no. AF160279, from which it differs at least 13/641 nucleotides sequenced. This strain could represent a previously undescribed species. F. Verticillioides MRC 8169 and MRC 8170 were identified as F. Verticillioides on the basis of morphological characters. On the basis of AFLP fingerprint patterns, the two strains are clones (identity at 48/48 AFLP bands), which was not unexpected as these strains are duplicate subcultures of a single wild collection. The strains are cross-fertile (MAT-1) with standard tester strains of G. moniliformis. The AFLP fingerprints resemble those of one of the mating type tester strains of F. Verticillioides, KSU A-00149 (67% identity). On the basis of DNA sequences of portions of the genes encoding translation elongation factor 1R (GenBank no. AY662325), the ITS region of the ribosomal DNA repeat unit data (GenBank no. AY662326), and the gene encoding β-tubulin (GenBank no. AY665679), this strain also is F. Verticillioides. Production of Fumonisin B and Fumonisin C Analogues by Strains of F. Werticillioides. Tandem MS experiments resulted in successive losses of water and tricarboxyllic acid (TCA) groups from the alkyl backbone of each fumonisin analogue hence producing product ion mass spectra characteristic of the structural moieties present in the compounds (Table 2). The use of appropriate segments during the chromatographic run made scanning at optimum conditions possible for each of the analogues. FB1 and FC1, and FB3 and FC3, have similar retention times. The MS-MS experiment was thus undertaken so as to selectively excite the respective ions under a single segment, which provided for the robust detection and quantification of these closely eluting analytes. When cultured on maize, the fumonisin B analogues constituted 56-84% of the total fumonisins produced (Table 3). All of the F. Verticillioides strains (Table 3) produced FC1 (11410 mg/kg) and FC4 (8-570 mg/kg). Three strains, MRC 8169, MRC 8170, and MRC 4319 (Table 3), also produced FC3 (4-
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Table 1. Fusarium Strains Used for Fumonisin B and C Series Production Fusarium strain number MRCa
Korean
KSUb
received as
current identification
strain source
7547 8184 8171 8172 8169 8170 826 4315 4319 4321
CAR KCTC 16654 no. 4 no. 53 94-cob-01c 94-cob-01c
X-12910 X-12915 D-12913 X-12914 A-12911 A-12912
F. oxysporum F. oxysporum F. oxysporum F. oxysporum F. moniliforme F. moniliforme
F. oxysporum F. oxysporum F. proliferatum Fusarium sp. (undescribed) F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides
Korean, carnation wilt Korea, asparagus wilt Korea, sesame seed Korea, red bean Korea, corn Korea, corn Centane, South Africa, home-grown corn Centane, South Africa, home-grown corn Centane, South Africa, home-grown corn Centane, South Africa, home-grown corn
a Culture collection of the PROMEC Unit, Medical Research Council, Tygerberg, South Africa. b Culture collection of the Department of Plant Pathology, Kansas State University, Manhattan, Kansas. c Two single-spored cultures made from original Korean culture because of slight culture differences (sectors).
Table 2. Product Ions (m/z) of the Fumonisin B and C Series product ion
FB1
FB2
FB3
FC1
FC3
FC4
[M + H−H2O]+ [M + H−2H2O]+ [M + H−TCA]+ [M + H−H2O−TCA]+ [M + H−2TCA]+ [M + H−2TCA−H2O]+
704 686 546 528 370 352
688 670 530 512 354 336
688 670 530 512 354 336
690 672 532 514 356 338
674 656 516 498 340 322
658 640 500 482 324 306
Table 3. Production of Fumonisin Analogues by Korean and South African Fusarium Strains Fumonisin analogues (mg/kg)a
Fusarium strains F. verticillioides F. proliferatum Fusarium sp. F. oxysporum
MRC number 8169b 8170b 8171b 8172b 7547b 8184b
FC1
Korean Strains 15 170 n.d.c 9 150 130
South African Strains F. verticillioides 826 (batch 1)b 826 (batch 2)b 4315b 4319 (batch 1)b 4319 (batch 2)b 4321b 826d 4315d 4319d 4321d
92 35 11 410 120 17 12 13 73 6
FC3 FC4
FB1
FB2
FB3
4 48 n.d. n.d. n.d. n.d.
8 300 5 71 440 330
220 3800 130 2100 n.d. n.d.
25 630 14 1400 n.d. n.d.
30 530 6 110 n.d. n.d.
n.d. n.d. n.d. 33 10 n.d. n.d. n.d. 7 n.d.
230 120 63 570 150 69 n.d. n.d. 67 n.d.
2100 1400 260 5900 2700 360 190 220 980 94
720 600 87 1800 740 77 59 64 340 17
170 86 12 650 190 49 19 22 71 14
a
Each sample was analyzed in triplicate. b Cultured on corn patties. c n.d.: not detected (
